PROJECT SUMMARY Many adult-onset pathologies, including aging-associated cancers, fibrosis, and degenerative diseases, start with transient cellular dysfunction that, over long timescales, progresses to organ-scale, physiological dys- function. To develop effective and precise treatments, we must identify the cellular events that drive disease progress and pinpoint where and when these events occur. Live in vivo imaging is uniquely suited to study cell and tissue dynamics in their native context and in real time. However for internal organs, most imaging meth- odologies fall short of providing a continuous view of the cellular origins of disease; they track either discrete events over minutes to hours or longitudinal processes over weeks to months?but not both. ?Bellymount?, our new imaging platform for adult Drosophila, is poised to fill this gap. Bellymount?s sim- ple design enables individual cells to be visualized within all major abdominal organs in adult Drosophila. Since imaging is non-invasive, the same cells and organs can be visualized repeatedly over periods up to four weeks. This innovative technology enables the first longitudinal analyses of slow physiological phenomena in intact Drosophila adults. In the proposed work, we will develop a new long-term (?12 hour) time-lapse capability to augment Bellymount?s existing longitudinal capability, thus enabling multi-hour cellular events and multi-week physiologi- cal outcomes to be correlated directly in the same individuals. At present, animal viability limits Bellymount im- aging sessions to <2 hours, which is too short to fully capture multi-hour events and informative numbers of infrequent, minutes-long events. In Aim 1, we will extend imaging sessions toward a goal of ?12 hours. To prolong viability, we will incorporate microfluidic delivery of liquid nutrients and pulsed, attenuated anesthesia into the Bellymount platform. We will determine the duration of long-term time-lapse imaging that is compatible with viability and assess the frequency with which long-term imaging can be applied in the course of a longitu- dinal experiment. In Aim 2, we will provide proof-of-principle for our combined long-term and longitudinal meth- odology by constructing a full timeline of cellular events during acute intestinal injury and subsequent regenera- tion. We will record intestinal injury during acute ingestion of chemical toxins in real-time. Using the same ani- mals, we will longitudinally monitor subsequent regeneration for 2 weeks or until it is complete. Finally, we will compare the responses of young and old adults to gain insight into the key sources of ageing-associated re- generative decline. Overall, successful completion of these Aims will provide first-of-its-kind methodology for ultra-long-term imaging of cellular, organ-scale, and inter-organ physiology in adult Drosophila.